Tryptophan hydroxylase (EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)) in rat brainstem extracts is activated 2 to 2.5-fold by ATP and Mg ions in the presence of subsaturating concentrations of the cofactor, 6-methyltetrahydropterin (6MPH4). The activation requires Mg ions and ATP but is not dependent on either cAMP or cGMP. The effect of ATP and Mg ions on enzyme activity was enhanced by micromole concentrations of Ca ions and totally blocked by EGTA. Finally, removal of calmodulin from the brain extracts by affinity chromatography on a column of fluphenazine-Sepharose rendered tryptophan hydroxylase unresponsive to activation by ATP-Mg2 ion. The re-addition of calmodulin restored the ATP-Mg2 ion-induced activation only in the presence of Ca2 ion. Drugs which bind calmodulin in vitro also block the ATP-Mg2 ion effect on tryptophan hydroxylase.